Biology Reference
In-Depth Information
STEM
ROOT
PM Markers
1
2
3
1
2
3
(KDa)
200
150
100
75
50
37
25
20
15
10
Number of
bands
42
32
41
41
31
40
Fig. 2
One-DE of protein extracts obtained using different extraction protocols
from carnation stems and roots infected with
Fod
. Criterion Free Stain electro-
phoresis system Bio-Rad
®
was used. Extraction and precipitation with TCA (1);
TCA-acetone method (2); TCA-acetone-phenol method (3)
7. Take the gel image using Criterion Stain Free Gel Imaging
System (
see
Note 8
).
8. Evaluate the number of bands for each of the tested treat-
ments and in each plant organ under study, using the Quantity
One
®
software from Bio-Rad (Fig.
2
). Compare the obtained
results in number of bands and its resolution (
see
Note 9
).
3.4 Two-Dimensional
Gel Electrophoresis
on Short Strips
Perform 2-D electrophoresis of different amounts of protein in the
range of 100-200
g for 11 cm-long IPG strips (pH 3-10). The
isoelectric focusing (IEF) (fi rst dimension) is carry out in the
PROTEAN
®
IEF cell system. For the second dimension, pre-cast
gels can be used with the Criterion system (
see
Note 10
).
1. Add protein extract obtained from the tissue under study in
independent 1.5 mL tubes, in equivalent amounts of 100 and
200
μ
g of protein. Add IPG strip rehydration solution to a
fi nal volume of 185
μ
μ
L. Shake vigorously by vortexing (
see
Note 11
).
2. Pour gently the previously prepared mixture creating a line in
one of the rails of the IPG strip focusing tray, avoiding the
formation of bubbles.
3. Place an 11 cm-long IPG strip with a 3-10 pH range, ensur-
ing that it is properly placed to avoid the entry of air bubbles
between the strip and the sample. The position of the strip
must allow its end marks to coincide with the position of the
(+, anode) and (−, cathode) electrodes.
